Cre-Lox recombination - Wikipedia

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in ... Cre-Loxrecombination FromWikipedia,thefreeencyclopedia Jumptonavigation Jumptosearch Site-specificrecombinasetechnology Cre-Loxrecombinationisasite-specificrecombinasetechnology,usedtocarryoutdeletions,insertions,translocationsandinversionsatspecificsitesintheDNAofcells.ItallowstheDNAmodificationtobetargetedtoaspecificcelltypeorbetriggeredbyaspecificexternalstimulus.Itisimplementedbothineukaryoticandprokaryoticsystems.TheCre-loxrecombinationsystemhasbeenparticularlyusefultohelpneuroscientiststostudythebraininwhichcomplexcelltypesandneuralcircuitscometogethertogeneratecognitionandbehaviors.NIHBlueprintforNeuroscienceResearchhascreatedseveralhundredsofCredrivermouselineswhicharecurrentlyusedbytheworldwideneurosciencecommunity. Thesystemconsistsofasingleenzyme,Crerecombinase,thatrecombinesapairofshorttargetsequencescalledtheLoxsequences.Thissystemcanbeimplementedwithoutinsertinganyextrasupportingproteinsorsequences.TheCreenzymeandtheoriginalLoxsitecalledtheLoxPsequencearederivedfrombacteriophageP1. PlacingLoxsequencesappropriatelyallowsgenestobeactivated,repressed,orexchangedforothergenes.AtaDNAlevelmanytypesofmanipulationscanbecarriedout.TheactivityoftheCreenzymecanbecontrolledsothatitisexpressedinaparticularcelltypeortriggeredbyanexternalstimuluslikeachemicalsignaloraheatshock.ThesetargetedDNAchangesareusefulincelllineagetracingandwhenmutantsarelethalifexpressedglobally. TheCre-LoxsystemisverysimilarinactionandinusagetotheFLP-FRTrecombinationsystem.[1] Contents 1History 2Overview 2.1Crerecombinase 2.2loxPsite 3Hollidayjunctionsandhomologousrecombination 4Site-specificrecombination 4.1Mechanismofaction 4.2Efficiencyofaction 4.3Temporalcontrol 4.4Conditionalcelllineagetracing 5NaturalfunctionoftheCre-loxsystem 6ImplementationofmultipleloxPsitepairs 7Notesandreferences 8Externallinks History[edit] Cre-Loxrecombinationisaspecialtypeofsite-specificrecombinationdevelopedbyDr.BrianSauerandpatentedbyDuPontthatoperatedinbothmitoticandnon-mitoticcells,andwasinitiallyusedinactivatinggeneexpressioninmammaliancelllines.[2][3][4]Subsequently,researchersinthelaboratoryofDr.JameyMarthdemonstratedthatCre-LoxrecombinationcouldbeusedtodeleteloxP-flankedchromosomalDNAsequencesathighefficiencyinspecificdevelopingT-cellsoftransgenicanimals,withtheauthorsproposingthatthisapproachcouldbeusedtodefineendogenousgenefunctioninspecificcelltypes,indeliblymarkprogenitorsincellfatedeterminationstudies,inducespecificchromosomalrearrangementsforbiologicalanddiseasemodeling,anddeterminetherolesofearlygeneticlesionsindisease(andphenotype)maintenance.[5] Shortlythereafter,researchersinthelaboratoryofProf.KlausRajewskyreportedtheproductionofpluripotentembryonicstemcellsbearingatargetedloxP-flanked(floxed)DNApolymerasegene.[6]Combiningtheseadvancesincollaboration,thelaboratoriesofDrs.MarthandRajewskyreportedin1994thatCre-loxrecombinationcouldbeusedforconditionalgenetargeting.[7]Theyobserved≈50%oftheDNApolymerasebetagenewasdeletedinTcellsbasedonDNAblotting.ItwasunclearwhetheronlyonealleleineachT-cellor50%ofTcellshad100%deletioninbothalleles.ResearchershavesincereportedmoreefficientCre-LoxconditionalgenemutagenesisinthedevelopingTcellsbytheMarthlaboratoryin1995.[8]IncompletedeletionbyCrerecombinaseisnotuncommonincellswhentwocopiesoffloxedsequencesexist,andallowstheformationandstudyofchimerictissues.AllcelltypestestedinmicehavebeenshowntoundergotransgenicCrerecombination. Independently,JoeZ.TsienhaspioneeredtheuseofCre-loxPsystemforcelltype-andregion-specificgenemanipulationintheadultbrainwherehundredsofdistinctneurontypesmayexistandnearlyallneuronsintheadultbrainareknowntobepost-mitotic.[9]TsienandhiscolleaguesdemonstratedCre-mediatedrecombinationcanoccurinthepost-mitoticpyramidalneuronsintheadultmouseforebrain.[10] Thesedevelopmentshaveledtoawidespreaduseofconditionalmutagenesisinbiomedicalresearch,spanningmanydisciplinesinwhichitbecomesapowerfulplatformfordetermininggenefunctioninspecificcelltypesandatspecificdevelopmentaltimes.Inparticular,thecleardemonstrationofitsusefulnessinpreciselydefiningthecomplexrelationshipbetweenspecificcells/circuitsandbehaviorsforbrainresearch,[11]haspromotedtheNIHtoinitiatetheNIHBlueprintforNeuroscienceResearchCre-drivermouseprojectsinearly2000.[12][13]Todate,NIHBlueprintforNeuroscienceResearchCreprojectshavecreatedseveralhundredsofCredrivermouselineswhicharecurrentlyusedbytheworldwideneurosciencecommunity. Overview[edit] Cre-LoxrecombinationinvolvesthetargetingofaspecificsequenceofDNAandsplicingitwiththehelpofanenzymecalledCrerecombinase.Cre-Loxrecombinationiscommonlyusedtocircumventembryoniclethalitycausedbysystemicinactivationofmanygenes.[14][15]AsofFebruary2019,Cre–Loxrecombinationisapowerfultoolandisusedintransgenicanimalmodelingtolinkgenotypestophenotypes.[11][16][17] TheCre-loxsystemisusedasagenetictooltocontrolsitespecificrecombinationeventsingenomicDNA.Thissystemhasallowedresearcherstomanipulateavarietyofgeneticallymodifiedorganismstocontrolgeneexpression,deleteundesiredDNAsequencesandmodifychromosomearchitecture. TheCreproteinisasite-specificDNArecombinasethatcancatalysetherecombinationofDNAbetweenspecificsitesinaDNAmolecule.Thesesites,knownasloxPsequences,containspecificbindingsitesforCrethatsurroundadirectionalcoresequencewhererecombinationcanoccur. AdiagramdescribinghowLox71andLox66sitescanbeusedtocombinetwoplasmidsintoonecontiguousplasmid. WhencellsthathaveloxPsitesintheirgenomeexpressCre,arecombinationeventcanoccurbetweentheloxPsites.Crerecombinaseproteinsbindtothefirstandlast13bpregionsofaloxsiteformingadimer.Thisdimerthenbindstoadimeronanotherloxsitetoformatetramer.Loxsitesaredirectionalandthetwositesjoinedbythetetramerareparallelinorientation.ThedoublestrandedDNAiscutatbothloxPsitesbytheCreprotein.ThestrandsarethenrejoinedwithDNAligaseinaquickandefficientprocess.TheresultofrecombinationdependsontheorientationoftheloxPsites.Fortwoloxsitesonthesamechromosomearm,invertedloxPsiteswillcauseaninversionoftheinterveningDNA,whileadirectrepeatofloxPsiteswillcauseadeletionevent.IfloxPsitesareondifferentchromosomesitispossiblefortranslocationeventstobecatalysedbyCreinducedrecombination.Twoplasmidscanbejoinedusingthevariantloxsites71and66.[18] Crerecombinase[edit] TheCreprotein(encodedbythelocusoriginallynamedas"Causesrecombination",with"Cyclizationrecombinase"beingfoundinsomereferences)[19][20]consistsof4subunitsandtwodomains:Thelargercarboxyl(C-terminal)domain,andsmalleramino(N-terminal)domain.Thetotalproteinhas343aminoacids.TheCdomainissimilarinstructuretothedomainintheIntegrasefamilyofenzymesisolatedfromlambdaphage.Thisisalsothecatalyticsiteoftheenzyme. loxPsite[edit] loxP(locusofX-overP1)isasiteonthebacteriophageP1consistingof34bp.Thesiteincludesanasymmetric8bpsequence,variableexceptforthemiddletwobases,inbetweentwosetsofsymmetric,13bpsequences.Theexactsequenceisgivenbelow;'N'indicatesbaseswhichmayvary,andlowercaselettersindicatebasesthathavebeenmutatedfromthewild-type.The13bpsequencesarepalindromicbutthe8bpspacerisnot,thusgivingtheloxPsequenceacertaindirection.UsuallyloxPsitescomeinpairsforgeneticmanipulation.IfthetwoloxPsitesareinthesameorientation,thefloxedsequence(sequenceflankedbytwoloxPsites)isexcised;howeverifthetwoloxPsitesareintheoppositeorientation,thefloxedsequenceisinverted.Ifthereexistsafloxeddonorsequence,thedonorsequencecanbeswappedwiththeoriginalsequence.Thistechniqueiscalledrecombinase-mediatedcassetteexchangeandisaveryconvenientandtime-savingwayforgeneticmanipulation.Thecaveat,however,isthattherecombinationreactioncanhappenbackwards,renderingcassetteexchangeinefficient.Inaddition,sequenceexcisioncanhappenintransinsteadofainciscassetteexchangeevent.TheloxPmutantsarecreatedtoavoidtheseproblems.[21] 13bp 8bp 13bp ATAACTTCGTATA- NNNTANNN -TATACGAAGTTAT ExampleAlternateloxPSites[22] Name 13bpRecognitionRegion 8bpSpacerRegion 13bpRecognitionRegion Wild-Type ATAACTTCGTATA ATGTATGC TATACGAAGTTAT lox511 ATAACTTCGTATA ATGTATaC TATACGAAGTTAT lox5171 ATAACTTCGTATA ATGTgTaC TATACGAAGTTAT lox2272 ATAACTTCGTATA AaGTATcC TATACGAAGTTAT M2 ATAACTTCGTATA AgaaAcca TATACGAAGTTAT M3 ATAACTTCGTATA taaTACCA TATACGAAGTTAT M7 ATAACTTCGTATA AgaTAGAA TATACGAAGTTAT M11 ATAACTTCGTATA cgaTAcca TATACGAAGTTAT lox71 TACCGTTCGTATA NNNTANNN TATACGAAGTTAT lox66 ATAACTTCGTATA NNNTANNN TATACGAACGGTA loxPsym ATAACTTCGTATA atgtacat TATACGAACGGTA Hollidayjunctionsandhomologousrecombination[edit] Duringgeneticrecombination,aHollidayjunctionisformedbetweenthetwostrandsofDNAandadouble-strandedbreakinaDNAmoleculeleavesa3’OHendexposed.Thisreactionisaidedwiththeendonucleaseactivityofanenzyme.5’Phosphateendsareusuallythesubstratesforthisreaction,thusextended3’regionsremain.This3’OHgroupishighlyunstable,andthestrandonwhichitispresentmustfinditscomplement.SincehomologousrecombinationoccursafterDNAreplication,twostrandsofDNAareavailable,andthus,the3’OHgroupmustpairwithitscomplement,anditdoesso,withanintactstrandontheotherduplex.Now,onepointofcrossoverhasoccurred,whichiswhatiscalledaHollidayIntermediate. The3’OHendiselongated(thatis,basesareadded)withthehelpofDNAPolymerase.Thepairingofoppositestrandsiswhatconstitutesthecrossing-overorRecombinationevent,whichiscommontoalllivingorganisms,sincethegeneticmaterialononestrandofoneduplexhaspairedwithonestrandofanotherduplex,andhasbeenelongatedbyDNApolymerase.FurthercleavageofHollidayIntermediatesresultsinformationofHybridDNA. Thisfurthercleavageor‘resolvation’isdonebyaspecialgroupofenzymescalledResolvases.RuvCisjustoneoftheseResolvasesthathavebeenisolatedinbacteriaandyeast. Formanyyears,itwasthoughtthatwhentheHollidayjunctionintermediatewasformed,thebranchpointofthejunction(wherethestrandscrossover)wouldbelocatedatthefirstcleavagesite.Migrationofthebranchpointtothesecondcleavagesitewouldthensomehowtriggerthesecondhalfofthepathway.Thismodelprovidedconvenientexplanationforthestrictrequirementforhomologybetweenrecombiningsites,sincebranchmigrationwouldstallatamismatchandwouldnotallowthesecondstrandexchangetooccur.Inmorerecentyears,however,thisviewhasbeenchallenged,andmostofthecurrentmodelsforInt,Xer,andFlprecombinationinvolveonlylimitedbranchmigration(1–3basepairsoftheHollidayintermediate),coupledtoanisomerisationeventthatisresponsibleforswitchingthestrandcleavagespecificity. Site-specificrecombination[edit] Mainarticle:Site-specificrecombination Site-specificrecombination(SSR)involvesspecificsitesforthecatalyzingactionofspecialenzymescalledrecombinases.Cre,orcyclicrecombinase,isonesuchenzyme.Site-specificrecombinationis,thus,theenzyme-mediatedcleavageandligationoftwodefineddeoxynucleotidesequences. Anumberofconservedsite-specificrecombinationsystemshavebeendescribedinbothprokaryoticandeukaryoticorganisms.Ingeneral,thesesystemsuseoneormoreproteinsandactonuniqueasymmetricDNAsequences.Theproductsoftherecombinationeventdependontherelativeorientationoftheseasymmetricsequences.Manyotherproteinsapartfromtherecombinaseareinvolvedinregulatingthereaction.Duringsite-specificDNArecombination,whichbringsaboutgeneticrearrangementinprocessessuchasviralintegrationandexcisionandchromosomalsegregation,theserecombinaseenzymesrecognizespecificDNAsequencesandcatalysethereciprocalexchangeofDNAstrandsbetweenthesesites. Mechanismofaction[edit] AmodelexperimentingeneticsusingtheCre-loxsystem:theprematurestopsequencepresentinfloxedmiceisremovedonlyfromcellsthatexpressCrerecombinasewhenthemicearebredtogether Initiationofsite-specificrecombinationbeginswiththebindingofrecombinationproteinstotheirrespectiveDNAtargets.AseparaterecombinaserecognizesandbindstoeachoftworecombinationsitesontwodifferentDNAmoleculesorwithinthesameDNAstrand.AtthegivenspecificsiteontheDNA,thehydroxylgroupofthetyrosineintherecombinaseattacksaphosphategroupintheDNAbackboneusingadirecttransesterificationmechanism.ThisreactionlinkstherecombinaseproteintotheDNAviaaphospho-tyrosinelinkage.Thisconservestheenergyofthephosphodiesterbond,allowingthereactiontobereversedwithouttheinvolvementofahigh-energycofactor. Cleavageontheotherstrandalsocausesaphospho-tyrosinebondbetweenDNAandtheenzyme.AtbothoftheDNAduplexes,thebondingofthephosphategrouptotyrosineresiduesleavea3’OHgroupfreeintheDNAbackbone.Infact,theenzyme-DNAcomplexisanintermediatestage,whichisfollowedbytheligationofthe3’OHgroupofoneDNAstrandtothe5’phosphategroupoftheotherDNAstrand,whichiscovalentlybondedtothetyrosineresidue;thatis,thecovalentlinkagebetween5’endandtyrosineresidueisbroken.ThisreactionsynthesizestheHollidayjunctiondiscussedearlier. Inthisfashion,oppositeDNAstrandsarejoinedtogether.SubsequentcleavageandrejoiningcauseDNAstrandstoexchangetheirsegments.Protein-proteininteractionsdriveanddirectstrandexchange.Energyisnotcompromised,sincetheprotein-DNAlinkagemakesupforthelossofthephosphodiesterbond,whichoccurredduringcleavage. Site-specificrecombinationisalsoanimportantprocessthatviruses,suchasbacteriophages,adopttointegratetheirgeneticmaterialintotheinfectedhost.Thevirus,calledaprophageinsuchastate,accomplishesthisviaintegrationandexcision.Thepointswheretheintegrationandexcisionreactionsoccurarecalledtheattachment(att)sites.AnattPsiteonthephageexchangessegmentswithanattBsiteonthebacterialDNA.Thus,thesearesite-specific,occurringonlyattherespectiveattsites.Theintegraseclassofenzymescatalysethisparticularreaction. Efficiencyofaction[edit] TwofactorshavebeenshowntoaffecttheefficiencyofCre'sexcisionontheloxpair.First,thenucleotidesequenceidentityinthespacerregionofloxsite.EngineeredloxvariantswhichdifferonthespacerregiontendtohavevariedbutgenerallylowerrecombinationefficiencycomparedtowildtypeloxP,presumablythroughaffectingtheformationandresolutionofrecombinationintermediate.[23] AnotherfactoristhelengthofDNAbetweentheloxpair.IncreasingthelengthofDNAleadstodecreasedefficiencyofCre/loxrecombinationpossiblythroughregulatingthedynamicsofthereaction.[24][25][26]GeneticlocationofthefloxedsequenceaffectsrecombinationefficiencyaswellprobablybyinfluencingtheavailabilityofDNAbyCrerecombinase.[26]ThechoiceofCredriverisalsoimportantaslowexpressionofCrerecombinasetendstoresultinnon-parallelrecombination.Non-parallelrecombinationisespeciallyproblematicinafatemappingscenariowhereonerecombinationeventisdesignedtomanipulatethegeneunderstudyandtheotherrecombinationeventisnecessaryforactivatingareportergene(usuallyencodingafluorescentprotein)forcelllineagetracing.[26]Failuretoactivatebothrecombinationeventssimultaneouslyconfoundstheinterpretationofcellfatemappingresults. Temporalcontrol[edit] InducibleCreactivationisachievedusingCreER(estrogenreceptor)variant,whichisonlyactivatedafterdeliveryoftamoxifen.[27]ThisisdonethroughthefusionofamutatedligandbindingdomainoftheestrogenreceptortotheCrerecombinase,resultinginCrebecomingspecificallyactivatedbytamoxifen.Intheabsenceoftamoxifen,CreERwillresultintheshuttlingofthemutatedrecombinaseintothecytoplasm.Theproteinwillstayinthislocationinitsinactivatedstateuntiltamoxifenisgiven.Oncetamoxifenisintroduced,itismetabolizedinto4-hydroxytamoxifen,whichthenbindstotheERandresultsinthetranslocationoftheCreERintothenucleus,whereitisthenabletocleavetheloxsites.[28]Importantly,sometimesfluorescentreporterscanbeactivatedintheabsenceoftamoxifen,duetoleakageofafewCrerecombinasemoleculesintothenucleuswhich,incombinationwithverysensitivereporters,resultsinunintendedcelllabelling.[29]CreER(T2)wasdevelopedtominimizetamoxifen-independentrecombinationandmaximizetamoxifen-sensitivity. Conditionalcelllineagetracing[edit] Cellsaltertheirphenotypeinresponsetonumerousenvironmentalstimuliandcanlosetheexpressionofgenestypicallyusedtomarktheiridentity,makingitdifficulttoresearchthecontributionofcertaincelltypestodisease.Therefore,researchersoftenusetransgenicmiceexpressingCreERt2recombinaseinducedbytamoxifenadministration,underthecontrolofapromoterofagenethatmarksthespecificcelltypeofinterest,withaCre-dependentfluorescentproteinreporter.TheCrerecombinaseisfusedtoamutantformoftheoestrogenreceptor,whichbindsthesyntheticoestrogen4-hydroxytamoxifeninsteadofitsnaturalligand17β-estradiol.CreER(T2)resideswithinthecytoplasmandcanonlytranslocatetothenucleusfollowingtamoxifenadministration,allowingtighttemporalcontrolofrecombination.Thefluorescentreportercassettewillcontainapromotertopermithighexpressionofthefluorescenttransgenereporter(e.g.aCAGpromoter)andaloxPflankedstopcassette,ensuringtheexpressionofthetransgeneisCre-recombinasedependentandthereportersequence.UponCredrivenrecombination,thestopcassetteisexcised,allowingreportergenestoexpressspecificallyincellsinwhichtheCreexpressionisbeingdrivenbythecell-specificmarkerpromoter.Sinceremovalofthestopcassetteispermanent,thereportergenesareexpressedinalltheprogenyproducedbytheinitialcellswheretheCrewasonceactivated.Suchconditionallineagetracinghasprovedtobeextremelyusefultoefficientlyandspecificallyidentifyvascularsmoothmusclecells(VSMCs)andVSMC-derivedcellsandhasbeenusedtotesteffectsonVSMCandVSMC-derivedcellsinvivo.[30][31][32][33][34][35] NaturalfunctionoftheCre-loxsystem[edit] TheP1phageisatemperatephagethatcauseseitheralysogenicorlyticcyclewhenitinfectsabacterium.Initslyticstate,onceitsviralgenomeisinjectedintothehostcell,viralproteinsareproduced,virionsareassembled,andthehostcellislysedtoreleasethephages,continuingthecycle.Inthelysogeniccyclethephagegenomereplicateswiththerestofthebacterialgenomeandistransmittedtodaughtercellsateachsubsequentcelldivision.ItcantransitiontothelyticcyclebyalatereventsuchasUVradiationorstarvation. PhageslikethelambdaphageusetheirsitespecificrecombinasestointegratetheirDNAintothehostgenomeduringlysogeny.P1phageDNAontheotherhand,existsasaplasmidinthehost.TheCre-loxsystemservesseveralfunctionsinthephage:itcircularizesthephageDNAintoaplasmid,separatesinterlinkedplasmidringssotheyarepassedtobothdaughterbacteriaequallyandmayhelpmaintaincopynumbersthroughanalternativemeansofreplication.[36] TheP1phageDNAwhenreleasedintothehostfromthevirionisintheformofalineardoublestrandedDNAmolecule.TheCreenzymetargetsloxPsitesattheendsofthismoleculeandcyclisesthegenome.ThiscanalsotakeplaceintheabsenceoftheCreloxsystem[37]withthehelpofotherbacterialandviralproteins.TheP1plasmidisrelativelylarge(≈90Kbp)andhenceexistsinalowcopynumber-usuallyonepercell.Ifthetwodaughterplasmidsgetinterlinkedoneofthedaughtercellsofthehostwilllosetheplasmid.TheCre-loxrecombinationsystempreventsthesesituationsbyunlinkingtheringsofDNAbycarryingouttworecombinationevents(linkedrings->singlefusedring->twounlinkedrings).Itisalsoproposedthatrollingcirclereplicationfollowedbyrecombinationwillallowtheplasmidtoincreaseitscopynumberwhencertainregulators(repA)arelimiting.[36] ImplementationofmultipleloxPsitepairs[edit] MultiplevariantsofloxP,[38]inparticularlox2272andloxN,havebeenusedbyresearcherswiththecombinationofdifferentCreactions(transientorconstitutive)tocreatea"Brainbow"systemthatallowsmulti-colouringofmice'sbrainwithfourfluorescentproteins. 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